An Ontology for Facilitating Discussion of Catalytic Strategies of RNA-Cleaving Enzymes

Abstract

A predictive understanding of the mechanisms of RNA cleavage is important for the design of emerging technology built from biological and synthetic molecules that have promise for new biochemical and medicinal applications. Over the past 15 years, RNA cleavage reactions involving 2'-O-transphosphorylation have been discussed using a simplified framework introduced by Breaker that consists of four fundamental catalytic strategies (designated α, β, γ, and δ) that contribute to rate enhancement. As more detailed mechanistic data emerge, there is need for the framework to evolve and keep pace. We develop an ontology for discussion of strategies of enzymes that catalyze RNA cleavage via 2'-O-transphosphorylation that stratifies Breaker's framework into primary (1°), secondary (2°), and tertiary (3°) contributions to enable more precise interpretation of mechanism in the context of structure and bonding. Further, we point out instances where atomic-level changes give rise to changes in more than one catalytic contribution, a phenomenon we refer to as "functional blurring". We hope that this ontology will help clarify our conversations and pave the path forward toward a consensus view of these fundamental and fascinating mechanisms. The insight gained will deepen our understanding of RNA cleavage reactions catalyzed by natural protein and RNA enzymes, as well as aid in the design of new engineered DNA and synthetic enzymes.

Figure 1.

2′-O-transphosphorylation leading to cleavage of the RNA backbone (left of central arrow) and idealized transition state highlighting the general catalytic strategies (right of central arrow). - Arrangement of the O2′ nucleophile, P (scissile phosphorus), and O5′ leaving group in an in-line attack geometry (facilitated by contacts, indicated by blue arcs, that splay the N−1 and N+1 bases). - Stabilization (neutralization/protonation) of the negative charge accumulation on the non-bridging phosphoryl oxygens (NPOs). - Activation (deprotonation) of the O2′ nucleophile. - Stabilization (neutralization/protonation) of the accumulating negative charge on the O5′ leaving group. Although this schematic uses a transition state model to illustrate the fundamental catalytic strategies, these strategies can impact any state along the reaction coordinate. Colored ovals highlight each strategy and encompass the primary atomic positions (defined in section 4 below) associated with the chemical space of bonds for each strategy.

Figure 2.

Application of ontology in describing the effects of G33I mutation and NPO thio substitutions of the substrate in the twister ribozyme (Twr). Summarized in the center are key interactions (1–4) that are highlighted in each variant/substrate by ovals in colors corresponding to their associated catalytic strategies, γ (red) and β (green). The wild-type (WT) Twr is shown in the top left, G33I mutant (top right), S(R_P) substrate (bottom left), and G33I/S(R_P) (bottom right). Arrows indicate transformations between the different variant/substrates. Relative kinetic rates and catalytic effects resulting from each transformation are indicated with their associated arrows. Lack of color highlighting indicates an absence (or significant weakening) of an interaction present in the wild-type and has a normal catalytic effect, whereas emphasis of color/size of highlighting indicates an inverse effect (e.g., a larger red sphere at G33:N1 (1) indicates a favorable down-shifted pK_a).